The ultimate goal of the proposal presented herein is to use E. coli as whole cell biocatalysts for the production of a wide variety of TDP-deoxysugars including di- and tri-deoxysugars, amino sugars and branched-chain sugars. These specialized activated hexoses are found as important structural components throughout plant and microbial secondary metabolites often playing a crucial role in conferring activity in bioactive natural products such as antibiotics and anticancer therapeutics. In Phase I, we proposed to investigate a novel approach to produce rare TDP-deoxysugars in Escherichia coli through metabolic engineering. During the Phase I study we successfully modified E. coli AB707 to accumulate TDP-4-keto-6-deoxy-D-glucose (TKDG; a key intermediate of TDP-deoxysugars) through inactivations and overexpression of specific pathway enzymes. Using exogenous TDP-deoxysugar biosynthetic enzymes, the accumulated TKDG pools were converted to specific TDP-deoxysugars in yields up to 80 mg/L, exceeding our Phase I goal. The success of the feasibility study will be expanded in Phase II. Specifically, we will further increase TDP- deoxysugar yields through additional genetic engineering experiments to limit loss of glucose- -phosphate to competing pathways, increase thymidine triphosphates (TTP) pools, reduce catabolite repression by glucose, and optimize fermentation conditions. We will also expand our range of TDP-deoxysugar products by cloning and expressing several exogenous sugar biosynthetic genes for the production of 0 additional complex TDP-deoxysugars. In Phase III we will commercialize the technology by carrying out Partnered R&D programs with strategic partners to make new glycoconjugates, by offering TDP-deoxysugars as well as deoxysugars as research reagents, and by licensing the technology on an application-specific basis.